The bacteriophage T7 protein kinase enhances T7 growth under suboptimal growth conditions, including elevated temperature or limiting carbon source. T7PK phosphorylates numerous E. coli proteins, and it has been proposed that phosphorylation of these proteins is responsible for supporting T7 replication under stressful growth conditions. How the phosphorylation of host proteins supports T7 growth is not understood. Escherichia coli (Ec) RNase III is phosphorylated on serine in bacteriophage T7-infected cells. Phosphorylation of Ec-RNase III induces a ~4-fold increase in catalytic activity in vitro. Ec-RNase III is involved in the maturation of several T7 mRNAs, and it has been shown that RNase III processing controls the translational activity and stability of the T7 mRNAs. Perhaps T7PK phosphorylation of Ec- RNase III ensures optimal processing of T7 mRNAs under suboptimal growth conditions. In this study a biochemical analysis was performed on the N-terminal portion of the 0.7 gene (T7PK), exhibiting only the protein kinase activity. In addition to phosphotransferase activity, T7PK also undergoes self-phosphorylation on serine, which down-regulates catalytic activity by an unknown mechanism. Mass spectral analysis revealed that Ser216 is the autophosphorylation site in T7PK. The serine residue is highly conserved, which in turn suggests that autophosphorylation is a conserved reaction with functional importance. Phosphorylated T7PK exhibits reduced phosphotransferase activity, compared to its dephosphorylated counterpart (dT7PK). The dT7PK exhibits enhanced ability to phosphorylate proteins, as well as undergo autophosphorylation. The mechanism by which autophosphorylation inhibits T7PK activity is unknown. An in vitro phosphorylation assay revealed that T7PK directly phosphorylates RNase III. Ec-RNase III processing activity is stimulated from two to ten-fold upon phosphorylation by the T7PK. The primary site of phosphorylation in RNase III is found to be Ser33, and Ser34 may act as the recognition determinant for T7PK. This was established by Ser →Ala mutations at the concerned site. The enhancement of catalytic activity is primarily due to a larger turnover number (kcat), with some additional contribution from a greater substrate binding affinity, as revealed by lower Km and K‟D values. Substrate cleavage assays under single turn over conditions established that the product release is the rate limiting step. Since there is no significant increase in the kcat as measured under single-turnover (enzyme excess) conditions, the increase in the kcat in the steady-state is due to enhancement of the product release step, and not due to an enhancement of the hydrolysis (chemical) step. / Chemistry